# Role of actin bundlers during enterocyte differentiation

> **NIH NIH R01** · VANDERBILT UNIVERSITY · 2024 · $431,768

## Abstract

SUMMARY
During differentiation, enterocytes build an extensive apical array of microvilli known as the brush border, which
serves to amplify the plasma membrane surface area available for nutrient absorption. An individual microvillus
is simple in structure, consisting of a supporting core bundle of ~25 actin filaments that protrudes from the apical
surface wrapped in membrane. In addition to serving as the sole site of nutrient uptake, brush border microvilli
also provide an anchoring point for the glycocalyx and regulate interactions with luminal microbes. Although the
brush border serves as the primary functional interface of the intestinal tract, mechanisms that drive the timely
formation of microvilli during enterocyte differentiation remained unclear until recently. During our first funding
period, we discovered several factors that control actin filament polymerization during microvilli formation,
including the IRTKS/EPS8 complex. However, building stable microvilli also requires that actin filaments are
organized into core bundles, which exhibit flexural rigidities high enough to deform the apical surface. How
nascent enterocytes coordinate the fundamental activities of actin filament polymerization and bundling in space
and time to build stable microvilli remains unknown. In recent preliminary studies, we used a proximity labeling
approach to identify proteins within ~20 nm of IRTKS/EPS8 puncta during microvillus assembly; this screen led
to our exciting discovery of Mitotic Spindle Positioning (MISP) as a new actin filament bundling protein in the
brush border. MISP is expressed along the full crypt-villus axis, where it localizes to the apical surface. Closer
inspection with super-resolution microscopy revealed that MISP exhibits strikingly specific enrichment on core
bundle rootlets. In cultured cells, we found that MISP stabilizes and elongates rootlets, and recruits other
canonical actin bundlers to these sites. Importantly, we found that purified MISP is sufficient to organize actin
filaments into tight linear bundles in vitro. Finally, our preliminary analysis of MISP knockout mice revealed a
striking loss of rootlets and decrease in microvillar surface density. Based on our preliminary data, we propose
the following CENTRAL HYPOTHESIS: At the apical surface of differentiating enterocytes, MISP organizes actin
filaments generated by the IRTKS/EPS8 complex to form core bundles that support the protrusion of brush
border microvilli. Using a unique combination of state-of-the-art light and electron microscopy technology and
novel biological model systems, we will: (Aim 1) determine if MISP specifies sites of microvillar growth at the
apical surface, (Aim 2) define the mechanism of MISP actin binding and bundling, (Aim 3) elucidate the function
of MISP in enterocyte differentiation in vivo. We expect that completion of these Aims will lead to new paradigms
for understanding intestinal epithelial morphogenesis.

## Key facts

- **NIH application ID:** 10861043
- **Project number:** 5R01DK111949-08
- **Recipient organization:** VANDERBILT UNIVERSITY
- **Principal Investigator:** MATTHEW J TYSKA
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $431,768
- **Award type:** 5
- **Project period:** 2017-02-01 → 2027-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10861043

## Citation

> US National Institutes of Health, RePORTER application 10861043, Role of actin bundlers during enterocyte differentiation (5R01DK111949-08). Retrieved via AI Analytics 2026-05-28 from https://api.ai-analytics.org/grant/nih/10861043. Licensed CC0.

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